Duplex-forming Ability Research Articles

MicroLOCK: Highly stable microgel biosensor using locked nucleic acids as bioreceptors for sensitive and selective detection of let-7a

Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2μL) at 25°C with a higher sensitivity (LOD 1.3fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N=7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo.

Synthesis, Duplex-Forming Ability, and Enzymatic Stability of Oligonucleotides Modified with Amide-Linked Dinucleotides Containing a 3',4'-Tetrahydropyran-Bridged Nucleic Acid.

Replacement of a phosphodiester linkage with an amide linkage can improve the binding affinity of oligonucleotides to complementary RNA and their stability toward nucleases. In addition, restricting the conformation of the sugar moiety and the phosphate backbone in oligonucleotides effectively improves duplex stability. In this study, we designed amide-linked dinucleotides containing a 3',4'-tetrahydropyran-bridged nucleic acid (3',4'-tpBNA) with a constrained sugar conformation as well as a torsion angle ε. Phosphoramidites of the designed dinucleotides were synthesized and incorporated into oligonucleotides. Conformational analysis of the synthesized dinucleotides showed that the sugar conformation of the S-isomer of the amide-linked dinucleotide containing 3',4'-tpBNA was N-type, which has the same conformation as that of the RNA duplex, while that of another R-isomer was S-type. Tm analysis indicated that the oligonucleotides containing the synthesized S-isomer showed RNA-selective hybridizing ability, although their duplex-forming ability was slightly inferior to that of natural oligonucleotides. Interestingly, the stability of the oligonucleotides toward endonucleases was significantly improved by modification with the two types of amide-linked dinucleotides developed in this study.

Post-synthetic nucleobase modification of oligonucleotides by Sonogashira coupling and influence of alkynyl modifications on the duplex-forming ability.

Recently, we reported that the alkynyl modification of nucleobases mitigates the toxicity of antisense oligonucleotides (ASO) while maintaining the efficacy. However, the general effect of alkynyl modifications on the duplex-forming ability of oligonucleotides (ONs) is unclear. In this study, post-synthetic nucleobase modification by Sonogashira coupling in aqueous medium was carried out to efficiently evaluate the physiological properties of various ONs with alkynyl-modified nucleobases. Although several undesired reactions, including nucleobase cyclization, were observed, various types of alkynyl-modified ONs were successfully obtained via Sonogashira coupling of ONs containing iodinated nucleobases. Evaluation of the stability of the duplex formed by the synthesized alkynyl-modified ONs showed that the alkynyl modification of pyrimidine was less tolerated than that of purine, although both the modifications occurred in the major groove of the duplex. These results can be attributed to the bond angle of the alkyne on the pyrimidine and the close proximity of the alkynyl substituents to the phosphodiester backbone. The synthetic method developed in this study may contribute to the screening of the optimal chemical modification of ASO because various alkynyl-modified ONs that are effective in reducing the toxicity of ASO can be easily synthesized by this method.

Structures of increased duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine-bridged nucleic acids

Structures of increased duplex-forming ability of oligonucleotides modified with <i>N</i>-<i>tert</i>-butylguanidine-bridged nucleic acids

Mechanism of the extremely high duplex-forming ability of oligonucleotides modified with N-tert-butylguanidine- or N-tert-butyl-N'- methylguanidine-bridged nucleic acids.

Antisense oligonucleotides (ASOs) are becoming a promising class of drugs for treating various diseases. Over the past few decades, many modified nucleic acids have been developed for application to ASOs, aiming to enhance their duplex-forming ability toward cognate mRNA and improve their stability against enzymatic degradations. Modulating the sugar conformation of nucleic acids by substituting an electron-withdrawing group at the 2'-position or incorporating a 2',4'-bridging structure is a common approach for enhancing duplex-forming ability. Here, we report on incorporating an N-tert-butylguanidinium group at the 2',4'-bridging structure, which greatly enhances duplex-forming ability because of its interactions with the minor groove. Our results indicated that hydrophobic substituents fitting the grooves of duplexes also have great potential to increase duplex-forming ability.

Synthesis and Properties of Nucleobase-Sugar Dual Modified Nucleic Acids: 2'-OMe-RNA and scpBNA Bearing a 5-Hydroxycytosine Nucleobase.

Naturally occurring 5-hydroxycytosine (5-OHCyt), which is associated with DNA damage, was recently found to reduce the hepatotoxicity of antisense oligonucleotides (ASOs) without compromising its antisense activity when used as a replacement for cytosine (Cyt). Additionally, sugar-modified nucleic acids, such as 2'-O-methylribonucleic acid (2'-OMe-RNA) and 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA), have emerged as useful antisense materials. Herein, we aimed to combine these two advantages by designing dual modified nucleic acids 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt bearing the 5-OHCyt nucleobase to develop efficient and safe ASOs. We describe the synthesis of 2'-OMe-RNA-5-OHCyt and scpBNA-5-OHCyt phosphoramidites and their incorporation into oligonucleotides (ONs). The duplex-forming ability and base discrimination properties of 2'-OMe-RNA-5-OHCyt- and scpBNA-5-OHCyt-modified ONs were similar to those of 2'-OMe-RNA-Cyt- and scpBNA-mCyt-modified ONs, respectively. We also synthesized two 2'-OMe-RNA-5-OHCyt-modified ASOs, and one of the two was found to exhibit reduced hepatotoxicity while retaining target mRNA knockdown activity in in vivo experiments.

Synthesis, Duplex-Forming Ability, and Nuclease Resistance of Oligonucleotides Containing a Thymidine Derivative with a 1-Oxaspiro[4.5]decane Skeleton.

Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6'-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6'-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6'-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6'-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6'-C-spiro-thymidine on the 3'-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.

Synthesis and duplex-forming ability of oligonucleotides modified with 4′-C,5′-C-methylene-bridged nucleic acid (4′,5′-BNA)

We describe herein the design and synthesis of 4′-C,5′-C-methylene-bridged nucleic acid (4′,5′-BNA), a novel artificial nucleic acid with the torsion angle γ in a non-canonical +ac range. The 4′,5′-BNA phosphoramidite bearing a thymine nucleobase was synthesized from a commercially available thymidine analog in 11 steps and successfully incorporated into oligonucleotides. The resulting oligonucleotides were evaluated for their duplex-forming ability toward single-stranded DNA and RNA.

2',4'-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance*.

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex‐forming abilities and enhanced nuclease resistance. 2′‐O,4′‐C‐Methylene‐bridged nucleic acid/locked nucleic acid (2′,4′‐BNA/LNA) with 1,3‐diaza‐2‐oxophenoxazine (BNAP (BH)) was previously reported. Herein, a novel BH analogue, 2′,4′‐BNA/LNA with 9‐(2‐aminoethoxy)‐1,3‐diaza‐2‐oxophenoxazine (G‐clamp), named BNAP‐AEO (BAEO), was designed. The BAEO nucleoside was successfully synthesized and incorporated into oligodeoxynucleotides (ODNs). ODNs containing BAEO possessed up to 104‐, 152‐, and 11‐fold higher binding affinities for complementary (c) RNA than those of ODNs containing 2′‐deoxycytidine (C), 2′,4′‐BNA/LNA with 5‐methylcytosine (L), or 2′‐deoxyribonucleoside with G‐clamp (PAEO), respectively. Moreover, duplexes formed by ODN bearing BAEO with cDNA and cRNA were thermally stable, even under molecular crowding conditions induced by the addition of polyethylene glycol. Furthermore, ODN bearing BAEO was more resistant to 3′‐exonuclease than ODNs with phosphorothioate linkages.

Synthesis and properties of GuNA purine/pyrimidine nucleosides and oligonucleotides.

We recently designed guanidine-bridged nucleic acids (GuNA), and GuNA bearing a thymine (T) nucleobase was synthesized and successfully incorporated into oligonucleotides. The GuNA-T-modified oligonucleotides possessed high duplex-forming ability towards their complementary single-stranded RNAs and were highly stable against 3'-exonuclease. Therefore, GuNA is a promissing artificial nucleic acid for therapeutic antisense oligonucleotides. We herein report the facile synthesis of GuNA phosphoramidites bearing adenine (A), guanine (G), and 5-methylcytosine (mC) nucleobases and a robust method for the preparation of GuNA-modified oligonucleotides, even with sequences having acid-sensitive purine nucleobases. Oligonucleotides modified with GuNA-A, -G, or -mC possessed high duplex-forming ability, similar to those modified with GuNA-T. Moreover, some of the GuNA-modified oligonucleotides were revealed to have high base discriminating ability compared with that of their natural counterparts. GuNA nucleosides exhibited no genotoxicity in bacterial reverse mutation assays. Thus, all GuNAs (GuNA-T, -A, -G, and -mC) are now available to be examined in therapeutic applications.

Synthesis and Properties of Oligonucleotides Having Ethynylphosphonate Linkages.

Ethynylphosphonate (EP)-linked thymidine dimers were synthesized via a palladium-catalyzed cross-coupling reaction and successfully incorporated into oligonucleotides. The oligonucleotides containing EP linkages appropriately formed a duplex with their complementary single-stranded RNA (ssRNA) and single-stranded DNA. The oligonucleotides containing both the EP linkages and 2'-O,4'-C-methylene-bridged nucleic acid/locked nucleic acid exhibited strong duplex-forming ability toward the complementary ssRNA. The EP-modified oligonucleotides exhibited higher exonuclease resistances than their natural counterparts. Moreover, one EP modification to a gapmer-type antisense oligonucleotide resulted in a switch of the cleavage site in the target ssRNA. Therefore, the EP modification can be applied for controlling the cleavage site in the RNase H-dependent mechanism.

Derivative Synthesis toward Enhancement of the Biophysical Properties of 2′,4′-Bridged Nucleic Acids

Over the past few decades, a number of modified oligonucleotides have been synthesized and evaluated their biophysical properties. Modified oligonucleotides having high enzymatic stability, high duplex-forming ability toward single-stranded RNA (ssRNA), high sequence specificity, and good pharmacokinetic profiles, are highly desirable for therapeutic applications. We previously demonstrated that oligonucleotides modified with 2′-O,4′-C-methylene bridged nucleic acid/locked nucleic acid (2′,4′-BNA/LNA) have extremely high duplex-forming ability toward ssRNA. In recent years, we have been focusing on developing further strengthened bridged nucleic acids, e.g. 2′,4′-BNA/LNA analogues showing high enzymatic stability, high sequence specificity, high efficacy, and low toxicity. We herein report several promising bridged nucleic acids developed recently for therapeutic applications, especially in antisense and splice-switching oligonucleotides.

Thioamide-Bridged Nucleic Acid (thioAmNA) Containing Thymine or 2-Thiothymine: Duplex-Forming Ability, Base Discrimination, and Enzymatic Stability.

Oligonucleotides containing bridged nucleic acids (BNAs) show high duplex-forming ability towards target single-stranded RNA, so many BNAs have been developed for antisense applications. Amide-bridged nucleic acids (AmNAs), which are BNA analogues bearing an amide bond at the bridge, exhibit high duplex-forming ability, enzymatic stability, and antisense activity; thus, the AmNA motif represents a promising BNA scaffold. The high enzymatic stability of the AmNA motif is presumably attributable to the bulky amide structure, because it inhibits the access of nucleases to the phosphodiester linkage. Here, to improve enzymatic stability further, we designed thioAmNAs: thioamide-bridged nucleotides that have a bulkier bridge structure than AmNA. The synthesis of thioAmNAs bearing either thymine (thioAmNA-T) or 2-thiothymine (thioAmNA-S2 T) bases was successful, and the obtained monomers were introduced into designed oligonucleotides without noticeable by-product generation. The thioAmNA-T- and thioAmNA-S2 T-modified oligonucleotides showed strong binding affinity toward complementary single-stranded RNA, with the thioAmNA-S2 T-modified oligonucleotide displaying excellent base-discrimination capability. Moreover, both thioAmNA-T and thioAmNA-S2 T endowed oligonucleotides with higher resistance to enzymatic degradation than AmNA-T. These results indicate that thioAmNAs are potentially useful chemical modifications for oligonucleotide-based therapeutics.

Effective syntheses of 2',4'-BNANC monomers bearing adenine, guanine, thymine, and 5-methylcytosine, and the properties of oligonucleotides fully modified with 2',4'-BNANC.

We efficiently synthesized 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNANC) monomers bearing the four nucleobases, guanine, adenine, thymine, and 5-methylcytosine and incorporated these monomers into oligonucleotides. Initially, we carried out the transglycosylation reaction on several 2'-O-substituted 5-methyluridines to evaluate the effects of 2'-substitutions on this reaction. Under the optimized conditions, purine nucleobases were successfully introduced, and 2',4'-BNANC monomers bearing adenine or guanine were obtained over several steps. In addition, the improved synthesis of the 2',4'-BNANC monomers bearing thymine or 5-methylcytosine was also achieved. The obtained 2',4'-BNANC monomers were subsequently incorporated into oligonucleotides and the duplex-forming abilities of the modified oligonucleotides were investigated. Duplexes containing 2',4'-BNANC monomers in both or either strands were found to possess excellent thermal stabilities.

Hybridization and Mismatch Discrimination Abilities of 2',4'-Bridged Nucleic Acids Bearing 2-Thiothymine or 2-Selenothymine Nucleobase.

Oligonucleotides modified with 2'- O,4'- C-spirocyclopropylene-bridged nucleic acid (scpBNA) exhibit excellent duplex-forming ability with their complementary single-stranded RNA (ssRNA). Here, we demonstrate that scpBNA bearing a 2-thiothymine (scpBNA-S2T) or 2-selenothymine (scpBNA-Se2T) nucleobase provides robust mismatch discrimination capabilities to oligonucleotides without compromising their high binding affinities toward the full complementary ssRNA. X-ray crystallographic analysis of a self-assembling oligonucleotide featuring 2',4'-BNA/LNA-2-thiothymine (2',4'-BNA/LNA-S2T, where 2',4'-BNA and LNA stand for "2'- O,4'- C-methylene-bridged nucleic acid" and "locked nucleic acid", respectively), a prototype of scpBNA-S2T, revealed that the 2-thiocarbonyl moiety plays a crucial role in the destabilization of thymine-guanine mismatched wobble base pairs.

2'-C,4'-C-Ethyleneoxy-Bridged 2'-Deoxyribonucleic Acids (EoDNAs) with Thymine Nucleobases: Synthesis, Duplex-Forming Ability, and Enzymatic Stability.

This chapter describes procedures for (1) the synthesis of six 2'-C,4'-C-ethyleneoxy-bridged thymidine phosphoramidites, i.e., methylene-EoDNA-T, (R)-Me-methylene-EoDNA-T, (S)-Me-methylene-EoDNA-T, EoDNA-T, (R)-Me-EoDNA-T, and (S)-Me-EoDNA-T phosphoramidites, (2) the introduction of the phosphoramidites into oligonucleotides, (3) UV-melting experiments of the duplexes of the modified oligonucleotides and complementary RNA, and (4) nuclease degradation experiments of the modified oligonucleotides.

2'-O,4'-C-Methylene-Bridged Nucleic Acids Stabilize Metal-Mediated Base Pairing in a DNA Duplex.

The 2'-O,4'-C-methylene-bridged or locked nucleic acid (2',4'-BNA/LNA), with an N-type sugar conformation, effectively improves duplex-forming ability. 2',4'-BNA/LNA is widely used to improve gene knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal-mediated base pairs (MMBPs), such as thymine (T)-HgII -T and cytosine (C)-AgI -C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2',4'-BNA/LNA in the field of MMBPs. 2',4'-BNA/LNA with 5-methylcytosine stabilized the MMBP of C with AgI ions. Moreover, the 2',4'-BNA/LNA sugar significantly improved the duplex-forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex-containing MMBPs. The results indicate that 2',4'-BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies.

Synthesis and hybridizing properties of isoDNAs including 3′-O,4′-C-ethyleneoxy-bridged 5-methyluridine derivatives

3′,4′-Ethyleneoxy-bridged 5-methyluridine derivatives with methyl groups in the bridge, (R)-Me-3′,4′-EoNA-T and (S)-Me-3′,4′-EoNA-T, were synthesized, and these two analogs and unsubstituted 3′,4′-EoNA-T were successfully incorporated into a 2′,5′-linked oligonucleotide (isoDNA). Their duplex-forming ability with complementary DNA and complementary RNA, and triplex-forming ability with double-stranded DNA, were evaluated by UV-melting experiments. The results indicated that isoDNAs, including these 3′,4′-EoNA analogs, could hybridize exclusively with complementary RNA. In particular, 3′,4′-EoNA-T and (R)-Me-3′,4′-EoNA-T modifications within isoDNA could stabilize the duplexes with complementary RNA compared with unmodified or 3′,4′-BNA-modified isoDNAs.

Facile synthesis and fundamental properties of an N-methylguanidine-bridged nucleic acid (GuNA[NMe

An N-methylguanidine-bridged nucleic acid (GuNA[NMe]), a guanidine-bridged nucleic acid (GuNA) bearing a methyl substituent at the bridge, was successfully synthesised and incorporated into oligonucleotides. By employing an acetyl protecting group, GuNA[NMe]-modified oligonucleotides bearing acid-sensitive purine nucleobases were successfully prepared. The obtained GuNA[NMe]-modified oligonucleotides exhibit excellent binding affinity towards the complementary single-stranded RNA and DNA. Furthermore, even a single GuNA[NMe] modification provides robust enzymatic stability, similar to that achieved by the well-established phosphorothioate backbone modification. These data indicate that such a GuNA[NMe] represents a valuable modification for the development of therapeutic oligonucleotides.

Synthesis, duplex-forming ability, enzymatic stability, and in vitro antisense potency of oligonucleotides including 2'-C,4'-C-ethyleneoxy-bridged thymidine derivatives.

We synthesized thymidine derivatives of 2'-C,4'-C-ethyleneoxy-bridged 2'-deoxyribonucleic acids with an 8'-methyl group ((R)-Me-EoDNA and (S)-Me-EoDNA) and without any substituent (EoDNA). Oligonucleotides including these EoDNAs showed high hybridization abilities with complementary RNA and excellent enzymatic stabilities compared with natural DNA. Moreover, the in vitro antisense potency of oligonucleotides with these EoDNAs and our recently reported methylene-EoDNAs was investigated and compared with that of LNA, which is a practical chemical modification for oligonucleotide-therapeutic agents. The results showed that EoDNAs and methylene-EoDNAs could be promising candidates for antisense technology.